Our interest is to understand how the DNA replication frequency is adjusted in the cell cycle. Our system is plasmid P1 whose replicon belongs to a family of replicons found in bacterial plasmids. The replication frequency in these replicons is controlled by short DNA repeat sequences (iterons). In the past year we have made significant progress in establishing that the iterons control replication by limiting initiator protein as well as by coupling ("handcuffing") replication origins via the bound initiators. These studies also showed that the plasmid paradigm is applicable to bacterial replicons. Regulation of Replication Frequency The P1 plasmid origin has five iterons that bind the plasmid-encoded initiator, RepA. It has been proposed that iterons control replication frequency by either limiting RepA or by handcuffing origins that causes steric hindrance to origin activity. To address the handcuffing model, we have developed an assay that involves comparison of plasmid copy numbers from cells that carry either plasmid monomer or isogenic plasmid dimer. Our premise is that communication (handcuffing) would occur more readily when the two origins are in cis, as in a dimer, because of higher local concentration of one site in the vicinity of another, than when they are in trans as in monomers. Dimer copy number was four-fold lower as compared to monomer in support of the handcuffing model. Evidence for direct physical interactions between origins was also obtained in vivo using a novel assay. Our studies provide the first physiological evidence that origin coupling can be an effective mechanism to reduce replication. The assays developed here can be applied to any protein, such as a transcription factor, with DNA looping activity. Initiator Mutants that Increase Plasmid Copy Number To establish the handcuffing model, we have isolated initiator mutants that increase the P1 plasmid copy number. These mutants, apparently defective in controlling plasmid overreplication, are being tested for handcuffing in vivo and in vitro. A strong correlation between copy number increase and the handcuffing defect will establish the importance of handcuffing in copy number control. If the handcuffing defect is found only in a subset of mutants, then presence of other modes of control can be argued. We are also developing new assays that can address handcuffing independently. Role of DnaA Boxes in the P1 Plasmid Origin The DnaA protein is essential for initiation of DNA replication in a wide variety of bacterial and plasmid replicons. The replication origin in these replicons invariably contains specific binding sites for the protein, called DnaA boxes. Plasmid P1 contains a set of DnaA boxes at each end of its origin but can function with either one of the sets. We found that the location of origin-opening, initiation site of replication forks, and directionality of replication do not change whether the boxes are present at both or at one of the ends of the origin. Replication was bidirectional in all cases. These results imply that DnaA functions similarly from the two ends of the origin. In spite of this dramatic flexibility, small changes in the box position (phasing) at either end of the origin significantly altered the origin efficiency. Most likely, the boxes not only help to increase local DnaA concentration but allow the protein to contact other components of the initiation complex in a phasing sensitive manner. A serendipitous observation was that sequences extraneous to the origin could greatly influence the origin activity. An IHF site was found in the extraneous sequence and the IHF protein was found to be essential for replication. We are currently studying the mechanism by which IHF could be activating the P1 origin. In a variety of DNA transactions IHF allows interactions between proteins bound to distant sites. We are pursuing a model where IHF is activating replication by bringing in distal DNA in close proximity of the initiation complex. The non-specific binding of the distal DNA to initiators could suffice to stabilize the complex. A similar mechanism has been suggested for the activation of transcription by DNA bending. Site-specific Binding of the Histone-like Protein, HU In addition to two initiators, DnaA and RepA, that bind to P1 origin at specific sites, initiation also requires HU. This protein is generally known to be a non-specific DNA binding protein. Recent studies in E. coli gal promoter and in phage Mu transposition have indicated that HU helps to form higher order nucleoprotein structures by site-specific binding. We have found that HU has higher affinity for the P1 origin compared to nonspecific DNA suggesting that HU may bind to P1 origin site-specifically. Footprinting studies in vivo were consistent with site-specific binding of HU in the region containing the iterons. HU also showed footprints specifically on iterons in vitro but the effect was weaker than in vivo. In supercoiled templates in vitro, HU repressed transcription in the same concentration range as was required to repress the gal promoter. How the site-specific binding of HU could help initiation remains to be understood.